228 - Evaluation of a Novel Mitochondria-targeted Peptide-based H2S Donor Compound (RTP-10) in Hyperglycaemia-induced Microvascular Endothelial Cell Dysfunction

Abstract

An overproduction of mitochondrial reactive oxygen species (ROS) in endothelial cells, is a major contributor to vascular endothelial dysfunction (VED) and angiopathy in diabetes. Hyperglycaemia (HG) induces metabolic changes in mitochondria, notably superoxide production, membrane hyperpolarisation and loss of ATP synthesis. Supplementation of cells and diabetic animals with sources of hydrogen sulfide (H2S) such as crude sulfide salts (e.g. NaSH) or the slow-release compound GYY4137 have been shown to protect the vasculature from the detrimental effects of oxidative stress (e.g. in hypoxia/ischaemia-reperfusion injury, diabetic angiopathy, stroke etc). However, since the major intracellular effect of H2S is on mitochondria (e.g. stimulation of cellular bioenergetics) massive concentrations (e.g. >200 µM) or doses in vivo (e.g. up to 1 g/kg for GYY4137) are required for this vascular protection as these approaches do not target H2S to where it is needed, i.e. the mitochondria. Two previously mitochondria-targeted H2S (mtH2SD) donor organic compounds based around a triphenylphosphonium (TPP+) targeting scaffold, AP39 and AP123 have been described in the literature which contain anethole dithiolethione and thiohydroxybenzamide as the H2S generating moieties. These compounds have shown significant efficacy when used therapeutically in several animal models of mitochondrial dysfunction at very low doses (7-300 µg / kg). Since the TPP+-based scaffolds require an active mitochondrial ΔΨm to accumulate within mitochondria, (a possible limitation) we now describe an alternative approach to target H2S to mitochondria using a novel H2S donor derivative of D-Arg-L-Tyr-L-Lys-L-Phe-NH2 (RTP10). We therefore exposed murine b.End3 brain microvascular endothelial cells to hyperglycaemia (HG) for 7 days to induce mitochondrial ‘stress’ and then added RTP10 (0.1 nM to 1 µM) for 3 days. After this time, the reversal of HG-induced metabolic changes, specifically mitochondrial ΔΨm (JC-1), mitochondrial superoxide, (mitosox) and ATP synthesis (by luminescence) were measured. RTP10 caused a concentration-dependent increase in mitochondrial H2S levels as well as reversed HG-induced mitochondrial hyperpolarisation and oxidant production, and restored ATP synthesis. Our study further suggests that targeting H2S to mitochondria may be a useful therapeutic strategy for reversing oxidative stress-induced mitochondrial damage in diabetes and other vascular and metabolic pathologies.